Erbb-2 selective small molecule kinase inhibitors

ABSTRACT

A novel method for erbB-2 kinase inhibition by compounds identifies through computational modeling and data processing and/or rational and de novo drug design is provided the compounds bind erbB-2 kinase molecules and which can be used as erbB-2 kinase agonists or antagonists. These compounds are useful especially in the treatment of cancer, particularly breast cancer, and can be used alone or in combination with other chemotherapeutic agents, particularly with hercetin, a humanized anti-HER-2 antibody, or with radiation therapy. A specific compound which is exemplified is “compound B17”=methyl-(posa-notrophenyl)-2-propynoate.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional PatentApplication Serial No. 60/221,515, filed Jul. 30, 2000, the entirety ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel method of prevention ortreatment of diseases where signal transduction pathways mediated byerbB-2 tyrosine kinase play a significant role. Examples thereof includeabnormal cell proliferation, including cancer, particularly, breastcancer.

BACKGROUND OF THE INVENTION

[0003] For mammalian cells to survive, they must be able to respondrapidly to changes in their environment. Furthermore, for cells toreproduce and carry out other cooperative functions, they must be ableto communicate efficiently with each other.

[0004] Cells most frequently adapt to their environment and communicatewith one another by means of chemical signals. An important feature ofthese signaling mechanisms is that in almost all cases a cell is able todetect a chemical signal without it being necessary for the chemicalmessenger itself to enter the cell. This permits the cell to maintainthe homeostasis of its internal environment, thereby permitting the cellto respond to its external environment without being adversely affectedby it.

[0005] These sensing functions are carried out by a variety ofreceptors, which are dispersed on the outer surface of the cell andfunction as “molecular antennae.” These receptors detect an incomingmessenger and activate a signal pathway that ultimately regulates acellular process such as secretion, contraction, metabolism or growth.

[0006] In the cell's cellular plasma membrane, transduction mechanismstranslate external signals into internal signals, which are then carriedthroughout the interior of the cell by chemicals known as “secondmessengers.”

[0007] In molecular terms, the process depends on a series of proteinswithin the cellular plasma membrane, each of which transmits informationby inducing a conformational change in the protein next in line. At somepoint, the information is assigned to small molecules or even to ionswithin the cell's cytoplasm, which serve as the above-mentioned secondmessengers. The diffusion of the second messengers enables a signal topropagate rapidly throughout the cell.

[0008] Abnormal cell signaling has been associated with cancer diseases.Cell signaling plays a crucial role in cell growth, proliferation anddifferentiation. Thus, when normal cell signaling pathways are altered,uncontrolled cell growth, proliferation and/or differentiation can takeplace, leading to the formation and propagation of cancer.

[0009] Cancer is the leading cause of death, second only to heartdisease in both men and women. Breast cancer is the most common tumor inwomen, representing 32% of all new cancer cases and causing 18% ofcancer-related deaths of women in the United States. In the fightagainst cancer, numerous techniques have been developed and are thesubject of current research to understand the nature and cause of thedisease, and to provide techniques for the control or cure thereof

[0010] One promising avenue for the development of cancer treatments isbased on blocking abnormal cell signaling pathways. Particular effortsare directed to the elucidation and regulation of the activity ofreceptor and trans-membrane proteins.

[0011] The human epidermal growth factor (EGF) is a six kilodalton(kDa), 53 amino acid, single-chain polypeptide which exerts itsbiological effect by binding to a specific 170 kDa cell membranereceptor (EGF-Rc). The human EGF-Rc consists of an extracellular domainwith a high cysteine content and N-linked glycosylation, a singletransmembrane domain, and a cytoplasmic domain with tyrosine kinaseactivity.

[0012] Many types of cancer display enhanced EGF-Rc expression on theircell surface membranes. Enhanced expression of the EGF-Rc can increasesignalling via receptor-mediator pathways which lead to pleiotropicbiological effects including excessive proliferation and metastasis.Examples include prostate cancer, breast cancer, lung cancer, head andneck cancer, bladder cancer, melanoma, and brain tumors.

[0013] In breast cancer, expression of the EGF-Rc is a significant andindependent indicator for recurrence and poor relapse-free survival. Theepidermal growth factor receptor (EGF-Rc) of cancer cells thereforerepresents a potential target for biotherapy.

[0014] EGFR and its physiologic ligands, epidermal growth factor (EGF)and transforming growth factor alpha (TGF alpha), play a prominent rolein the growth regulation of many normal and malignant cell types. Onerole the EGF receptor system may play in the oncogenic growth of cellsis through autocrine-stimulated growth. Cells which express EGFR andsecrete EGF and/or TGFalpha can stimulate their own growth, therebycreating a cancerous condition.

[0015] An autocrine growth stimulatory pathway analogous with thatproposed for epidermal growth factor receptor and its ligands may alsobe employed by a growing list of oncogene encoded transmembrane proteinsthat have a structure reminiscent of that of the growth factorreceptors.

[0016] The HER-2/neu or c-erbB-2 oncogene belongs to the erbB-likeoncogene group, and is related to, but distinct from EGFR. The ErbB-2gene encodes a 185 kD transmembrane glycoprotein that has partialhomology with other members of the EGFR family. The expressed proteinhas been suggested to be a growth factor receptor due to its structuralhomology with EGFR. However, known EGFR ligands, such as EGF or TGF.alpha do not bind to p¹⁸⁵-erbB-2.

[0017] The erbB-2 oncogene has been demonstrated to be implicated in anumber of human adenocarcinomas leading to elevated levels of expressionof the p¹⁸⁵ protein product. For example, the erbB-2 oncogene has beenfound to be amplified in breast, ovarian, gastric and even lungadenocarcinomas. Furthermore, the amplification of the c-erbB-2 oncogenehas been found in many cases to be a significant, if not the mostsignificant, predictor of both overall survival time and time to relapsein patients suffering from such forms of cancer. Carcinoma of the breastand ovary account for approximately one-third of all cancers occurringin women and together are responsible for approximately one-fourth ofcancer-related deaths in females.

[0018] Significantly, the c-erbB-2 oncogene has been found to beamplified in 25 to 30% of human primary breast cancers and it has beenassociated with a high risk of relapse and death. In breast cancers witherbB-2 overexpression abnormal cell proliferation is believed to becaused by extremely high tyrosine kinase activity and the resulting highlevel of signal transduction.

[0019] Overexpression of HER-2 has also been found to be associated withincreased resistance to chemotherapy or patients with elevated levels ofHER-2 respond poorly to many drugs. It is believed that decreasing thelevels of HER-2 will allow chemotherapeutic drugs to be more effective.Therefore, therapies targeted at erbB-2 have the great therapeuticpotential for the treatment of breast cancers.

[0020] In view of the above, the development of new and potentanti-breast cancer drugs and the design of treatment protocols directedat the regulation of erbB-2 activity is an exceptional focal point forresearch in the modem therapy of breast cancer. Drug targeting is aparticularly attractive approach for killing malignant cells, whenleaving normal tissue unharmed is achieved.

[0021] ErbB-2 is a clinically proven therapeutic target for breastcancer. Indeed, the recently completed phase M clinical trial ofanti-Her2 Herceptin provide evidence that systemic administration ofHerceptin, alone and in combination with cytotoxic chemotherapy inpatients with erbB-2 overexpressing prinary tumors, can increase thetime to recurrence and overall response rates in metastatic breastcancer. Herceptin is recognized as the first in what promises to-be awave of therapies attacking cancer at its genetic roots.

[0022] Certain limitations are associated with large moleculestrategies, including poor delivery, poor in vivo stability, possibleimmune response and high cost. Accordingly, it is highly desirable toprovide therapies based on small molecules targeted at interfering witherbB receptor-mediated signal transduction pathways (including erbB-2,erbB-3 and erbB4). Compared to therapies based on large drug molecules,such as therapeutic antibodies, small molecule drug therapies have anumber of advantages, including good oral availability and low cost.

[0023] A number of criteria should be considered in the development ofsmall molecule erbB-2 kinase inhibitors, including good potency,selectivity, cell permeability, bioavailability, appropriatepharmacokinetics and non-toxicity.

[0024] In breast cancers with erbB-2 overexpression, abnormal cellproliferation is caused by the extremely high tyrosine kinase activityand resulting high level of signal transduction. Drugs blocking thisextremely high erbB-2 tyrosine kinase activity could have the potentialto shut down signaling pathways mediated by erbB-2. Thus, erbB-2 kinaseinhibitors that are capable of entering the cell, blocking tyrosinekinase activity and shutting down the signal transduction pathwaymediated by erbB-2 may be used as potential therapeutic agents for thetreatment of breast cancer. Furthermore, it has been shown that tyrosinekinase inhibitors synergize with antibodies to EGFR to inhibit thegrowth of aquamous cell carcinoma in vivo. Thus, a specific erbB-2kinase inhibitor may also have synergistic effects with Herceptin in thetreatment of breast cancer.

[0025] The development of small molecule kinase inhibitors of the EGFRfamily of receptors tyrosine kinases has been so far focused on EGFRitself. Very potent and selective EGFR small molecule kinase inhibitorshave been reported and some EGFR small molecule kinase inhibitors haveadvanced to phase I/II clinical trials for the treatment of certaincancer forms. To date, very few kinase inhibitors selective for erbB-2were reported.

[0026] Therefore, it would be greatly beneficial if new therapies couldbe designed based on identified existing compounds, rationally modifiedcompounds and/or de novo designed compounds which are active as erbB-2kinase inhibitors. In particular, it would be helpful if therapies basedon compounds having improved selectivity, solubility and stability couldbe obtained.

SUMMARY AND OBJECTS OF THE INVENTION

[0027] It is an object of the invention to provide novel therapies basedon inhibiting in vivo the erbB-2 kinase signaling pathway.

[0028] It is a more specific object of the invention to provide noveltherapies that result in the inhibition of cell proliferation and/ordifferentiation and/or the promotion of cell apoptosis comprising theadministration of a compound that erbB-2 kinase related cell growthsignaling.

[0029] It is an even more specific object of the invention to providenovel therapies that result in the inhibition of cell proliferationand/or differentiation and/or promotion of cell apoptosis by theadministration of a compound having formulae (I) to (V):

[0030] In a preferred embodiment, such therapies will comprise treatmentof cancer and other neoplastic conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures illustrate some of the compounds of the invention,methods for identifying those compounds and results of in vitro and invivo biological test demonstrating the activity of illustrativecompounds according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0032] In cells transformed By erbB-2 overexpression, therapeutic agentsinhibiting erbB-2 kinase activity can interrupt the flow of signaltransduction mediated by erbB-2 receptor to the ras pathway and mayresult in the reversal of the cancer phenotype. Thus, one object of theinvention is to provide therapies based on inhibition erbB-2 kinaseactivity.

[0033] The present invention provides therapies based on compoundscapable of interfering with erbB-2 kinase activity. In one aspect, theinvention provides therapies based on existing compounds which areidentified through computational modeling as inhibitors of erbB-2 kinaseactivity. In another aspect, the invention provides novel compoundswhich are designed by rational design modification or existing compoundsor de novo to provide high activity and selectivity and therapies basedon these compounds.

[0034] In a first aspect, the present invention provides novel therapiesbased on existing compounds which are identified as potent and selectivesmall molecule inhibitors of erbB-2 kinase. The compounds are identifiedthrough structure-based three-dimensional (3D) database searching. Thecompounds identified through database searching are processed throughbiological tests to identify one or more lead compounds for clinicaltesting and/or rational drug design refinement.

[0035] Computationally predicting a compound's binding affinity to ahost protein involves utilizing the three dimensional (3-D) structuresof the host protein and the compound. The 3-D structure of the compoundis obtained from a database of chemical compounds. The 3-D structure ofthe host protein can also be obtained from a protein database.

[0036] The invention provides potent and erbB-2 specific kinaseinhibitors through a structure-based drug discovery approach. Themethodology employed in the discovery of erbB-2 kinase inhibitors isdisclosed in U.S. patent application Ser. No. 09/301,339, filed on Apr.29, 1999, the contents of which are hereby incorporated by reference intheir entirety. A flow chart for the methodology is shown in FIG. 1.

[0037] Briefly, in structure-based 3D-database searching for drugdiscovery, once the 3D structure of the target molecule (a receptor oran enzyme) is determined, large chemical databases containing the 3Dstructures of hundreds of thousands of structurally diverse syntheticcompounds and natural products are searched through computerizedmolecular docking to identify small molecules that can interacteffectively with the target or host molecule. In spite of the massiveincrease in the number of biological molecules whose 3-D structure hasbeen elucidated, the majority of proteins of known primary structure(amino acid sequence) do not have a known tertiary (or 3-D) structure.

[0038] For drug design involving target proteins of unknown tertiarystructure, a model structure can be constructed based on the knowntertiary structure of a protein which is homologous to the targetprotein. The structure of the homologous protein is used to construct atemplate structure of all or part of the target protein. The structureobtained through homology modeling provides a working structure forfurther refinement. The working structure for the protein not having aknown structure is obtained by refining the template structure.

[0039] In forming a template 3-D structure of the host protein, eachatom of the backbone of the target protein is assigned a positioncorresponding to the position of the equivalent backbone atom in thehomologous protein. Similarly, each atom of a side chain of the targetprotein having an equivalent side chain in the homologous protein isassigned the position corresponding to the position of the atom in theequivalent side chain of the homologous protein. The atom positions of aside chain not having an equivalent in the homologous protein aredetermined by constructing the side chain according to preferredinternal coordinates and attaching the side chain to the backbone of thehost protein.

[0040] The template structure thus obtained is refined by minimizing theinternal energy of the template protein. During the refinement, thepositions of the atoms of the side chains having no equivalents in thehomologous protein are adjusted while keeping the rest of the atoms ofthe template protein in a fixed position. This allows the atoms of theconstructed side chains to adapt their positions to the part of thetemplate structure determined by homology. The full template structureis then minimized (relaxed) by allowing all the atoms to move. Relaxingthe template 3-D structure of the protein eliminates unfavorablecontacts between the atoms of the protein and reduces the strain in thetemplate 3-D structure.

[0041] Based on the refined structure of the target protein, ahost-guest complex is formed by disposing a compound from a compounddatabase in a receptor site of the protein. The structure of thehost-guest complex is defined by the position occupied by each atom inthe complex in a three dimensional referential.

[0042] A geometry-fit group is formed by selecting the compounds whichcan be disposed in the target binding site without significantunfavorable overlap with the atoms of the protein. For each compound inthe geometry fit group, a predicted binding affinity to the receptorsite of the host protein is determined by minimizing an energy functiondescribing the interactions between the atoms of the compound and thoseof the protein. The mininization of the energy function is conducted bychanging the position of the compound such that a guest-host complexstructure corresponding to a minimum of the energy function is obtained.The compounds having the most favorable energy interaction with theatoms of the binding site are identified for optional furtherprocessing, for example through display and visual inspection ofcompound protein complexes to identify the most promising compoundcandidates.

[0043] The displayed complexes are visually examined to form a group ofcandidate compounds for in vitro testing. For example, the complexes areinspected for visual determination of the quality of docking of thecompound into the receptor site of the protein. Visual inspectionprovides an effective basis for identifying compounds for in vitrotesting.

[0044] After putative binding compounds have been identified, theability of such compounds to specifically bind to erbB-2 kinase isconfirmed in vitro and/or in vivo.

[0045] The potency and selectivity of potential erbB-2 kinase inhibitorsis evaluated in vitro with breast cancer cells overexpressing erbB-2(MDA-453) or EGFR (MDA-468), model cells (32D cells transfected withEGFR or erbB-2/erbB3, erbB-2/erbB-4, erbB-4), or NIH3T3 cellstransfected with EGFR, EGFR/erbB-2 or erbB-2). Potent and selectiveinhibitors are tested further in their ability to inhibitcolony-formation in soft-agarose. Compounds having good in vitroactivity are tested in vitro. Tumor bearing mice are treated withtherapy, based on the compounds and the effect on the tumor size isobserved. Compounds showing effective tumor reduction are then used inclinical trial protocols.

[0046] Computational Identification of Compounds Having Potential erbB-2Kinase Inhibitory Activity

[0047] To date, the experimental 3D structure (including the kinasedomain) of either erbB-2 or EGFR has not been determined. However, thestructures of the kinase domain of a number of other receptor tyrosinekinases have been determined through X-ray crystallography. The kinasedomain of these receptor tyrosine kinases is closely related to those oferbB-2 and EGFR, which provides an opportunity to model the 3D structureof the kinase domain of erbB-2 and EGFR using the homology modelingapproach described above.

[0048] Protein kinases, including erbB-2 and EGFR, have an active and aninactive conformation. Inhibition of either of these two conformationalstates can lead to the inhibition of kinase activity.

[0049] The sequences (or primary structures) of erbB-2 and EGFR wereobtained from the Protein Gene Bank. Templates for homology modelingwere obtained by searching the Protein Databank. 3D structures of thereceptors were built using the homology-modeling based on the X-raystructure of the active and inactive insulin receptor tyrosine kinase asa template to model the active and inactive conformation of the erbB-2and EGFR kinase domains, respectively.

[0050] Insulin receptor tyrosine kinase domain has 35% identities, 52%similarities and 10% gaps when compared to that of erbB-2, and 35%identities, 52% similarities and 5% gaps when compared to that of EGFR.

[0051] In forming a template structure of the erbB-2 kinase receptordomain, each atom in the backbone of the erbB-2 kinase domain wasassigned a position corresponding to the position of the equivalent atomin the 3-D structure of the insulin receptor kinase domain. Similarly,each atom of a side chain of the erbB-2 kinase domain having anequivalent side chain in the insulin receptor kinase domain was assigneda position corresponding to the position of the atom in the equivalentside chain of the insulin receptor kinase domain. The atoms of the sidechains of the erbB-2 kinase domain not having equivalents in the insulinreceptor kinase domain were determined by positioning the side chainaccording to its position in the amino acid sequence of ErbB-2 kinaseand refining the template structure thus obtained. The refined templatestructure was then relaxed to reduce the strains which may have beenpresent in the refined template.

[0052] The backbone-3D structures of the activated insulin receptortyrosine kinase domain and the corresponding model structures of erbB-2and EGFR are shown in FIG. 2.

[0053] The ATP binding site of the erbB-2 kinase domain from the refinedstructure (both the active and inactive conformation) was used as thetarget for structure-based database searching.

[0054] Database searching was conducted by processing two databases, theNational Cancer Institute 3D-database (279,000 compounds) and theAvailable Chemical Database (250,000 compounds). Each compound in thedatabases was processed to identify compounds having a shape which iscomplementary to the shape of the erbB-2 ATP binding site. For eachcompound, a rigid body docking minimum energy was evaluated and thecompounds were ranked according to their rigid body docking energy. Inthis procedure each compound was rigidly docked into the modeled erbB-2ATP active site. That is, the compound was docked into the active sitewithout changing the internal coordinates of the compound.

[0055] A geometry fit/rigid body docking group was formed by the top20,000 compounds obtained through shape complementarity ranking. Thesecompounds were further processed by evaluating their energetic bindingaffinity the ATP binding site of erbB-2 kinase. The compounds wereprocessed through flexible docking processing. That is, the compoundswere allowed to adjust their internal coordinates during docking therebyallowing for flexible docking. Flexible docking allowed a more accuratedetermination of the energy of interaction between each compound and theatoms forming the ATP active site.

[0056] The top 2,000 compounds according to the flexible docking energyranking were further examined to eliminate highly charged compoundswhose ability to enter the cell would be greatly hampered by theirchange. Of the 2000 compounds selected through flexible docking, 1000compounds were selected for biological processing through in vitro andin vivo testing based on their net electrostatic change.

[0057] The potency and selectivity of potential erbB-2 kinase inhibitorswas evaluated in vitro with breast cancer cells overexpressing erbB-2(MDA-453) or EGFR (MDA-468) and model cells (32D cells transfected withEGFR or erbB-2/erbB3, erbB-2/erbB-4, erbB-4, or NIH3T3 cells transfectedwith EGFR, EGFR/erbB-2 or erbB-2).

[0058] The compounds were also tested for inhibitory activities inphosphorylation, cell growth and MAP kinase. Selected compounds werefurther tested for their ability to inhibit colony-formation insoft-agarose.

[0059] Cell proliferation assays were also performed. A solubletetrazolium/formazan (XTT) assay was performed to directly measure thecell killing activity in a 96-well plate. The soft-agar colony formationassay was employed to directly measure the transforming ability ofselect compounds as this test provides data that has been shown tocorrelate well with in vivo tumorigenicity.

[0060] In order to show the presence of erbB receptors in the biologicalmaterials used in testing the compounds identified or designed accordingto the invention, a series of antibodies specific for each of the erbBreceptors were tested for both western blotting and immunoprecipitationexperiments. Those antibodies were utilized to screen the expression oferbB receptors by western blot analysis in various breast cell fines aswell as others that over express the erbB receptors. Each receptor wasdetected with a specific antibody, e.g., EGFR was detected with mAb(UBI), erbB-2 was detected with mAb (Oncogene Sciences), erbB-3 wasdetected with mAb (Oncogene Sciences), and antiphosphotyrosine mAb (UBI)and visualized with ECL (Amersham). The results for these probing testsare summarized in Table I: TABLE I Autophos- Origin of phorylation atTumori- Cell Line Cells EGFR erbB-2 erbB-3 erbB-4 p¹⁸⁵ or p¹⁷⁰ genicityMDA-453 breast − +++ ++ +++ +++ +/− SKBr3 breast + ++++ ++ +/− BT-474breast +/− ++++ +++ +++ ++++ +(E2) NMA-361 breast − +++ ++ ++ +++ +(E2)MDA-468 breast ++++ − + n.d. +++ + MDA-231 breast ++ +/− − − + ++++ A431epidermal +++++ + ++ +/− ++++ ++++ MDA-435 breast. n.d. − ++ − − ++++MCF-7 breast +/− + ++ + − +(E2) N87 gastric − +++++ +++ ++++ +++ +++SKOV3 ovarian − +++++ +/− − ++ +++

[0061]FIG. 3 illustrates in vitro testing using 32D model cells. Thereare two main advantages of the 32D model cell system. First, the 32Dcells are devoid of many receptors, therefore, they provide almost zerobackground of receptor autophosphorylation or cross-talks betweenreceptors. 32D cells from non-tumorigenic, murine hematopoietic celllines are devoid of receptors for many growth factors (e.g., EGF, PDGF,erbB-2/314. KGF, IL-2, CSF-1, Met, Kit, etc.). Second, when 32D cellsare transfected with a particular growth factor receptor, dualniitogenic and signal transduction pathways are created for the sametransfectants expressing that receptor. For instance, 32D cellstransfected with erbB-4 will proliferate in the presence of either HRGor IL-3. This IL-3 dependence, however, can be bypassed by thestimulation of signal transduction pathways initiated by the expressionof specific growth factor receptors and the addition of the appropriateligand to the culture medium.

[0062] In investigating ligand-induced phosphorylation of the erbB-2receptor, we employed the 32D cells transfected with a combination oferbB-2 and erbB-3, since there is no binding or activation of erbB-2 insingle erbB-2 transfected 32D cells. In addition, the NIH 3T3 cellstransfected with the chimeric EGFR/erbB-2 receptor were used to testligand-induced erbB-2 phosphorylation. To investigate ligand-inducedphosphorylation of the EGFR receptor, 32D and NIH3T3 cells transfectedwith EGFR were used.

[0063] To investigate the inhibition of autophosphorylation of theerbB-2 receptor kinase, 32D and NIH 3T3 cells transfected with mutanterbB-2, the neu oncogenes, which exhibit a high level ofautophosphorylation as employed.

[0064] Furthermore, using 32D cells transfected with a single (EGFR,mutant erbB-2, erbB-4) or double receptors (EGFR/erbB-2, erbB-2/erbB-3,erbB-2/erbB-4, EGFR/erbB-3, EGFR/erbB-4), allowed for the determinationof preferential inhibitory activity blocking the kinase activityassociated with either the homodimers or the heterodimers. NIH3T3 andMCF-7 transfected with other receptor tyrosine kinases including FGFR,PDGFR, VEGFR or ras were employed to evaluate their selectivity overreceptor kinases not related with the EGFR family.

[0065] 141 of the compounds identified through computational processingwere tested for their ability to inhibit phosphorylation in human breastcancer cell line MDA-453 that overexpresses erbB-2 obtained by geneamplification. Ten compounds were found to inhibit >90% of theauto-phosphorylation activity of erbB-2 at 100_M. The ten compounds werefurther tested in a dose-dependent phosphorylation assay both in anMDA-453 cell line, and an MDA-468 cell line. The latter overexpressesthe EGFR receptor.

[0066] Of the ten compounds identified above, six compounds were foundto have relative selectivity in inhibiting phosphorylation in MDA-453cells over MDA-468 cells. One particular compound that was found to haveexcellent biological activity is B17. FIG. 4(A) shows the structure ofcompound B17 and FIG. 4(B) shows compound B17 within the modeled activesite of erbB-2 kinase.

[0067]FIG. 5(C) shows the irreversible inhibition obtained, by treatingthe cells with compound B17 for 30 min and washout for 8 hours, thenassaying for tyrosine phospohrylation. As shown in FIGS. 5(A) to 5(C),lead compound B17 has excellent potency (IC₅₀ was estimated to beapproximately 1-2_M) and does not inhibit the EGFR phosphorylation in upto 400_M. The results shown in FIG. 5(A) to 5(C) show, unexpectedly thatlead compound B17 has a selectivity more than 100-fold for erbB-2 overEGFR.

[0068] B17 exposure did not affect the expression of erbB-2 or EGFR.Moreover, using P32 labeled ATP binding assay, we confirmed that B17blocks the binding of ATP to erbB-2 but not to EGFR, suggesting that B17is indeed an ATP competitive inhibitor.

[0069] In further probing the biological activity of B17, the NIH-3T3cells that overexpress either EGFR, erbB-2 or-the chimeric EGFR(extracellular) and erbB-2 (intracellular) receptor through transfectionwere exposed to treatmetn with B17. Both EGFR and chimeric EGFR/erbB-2depend on the addition of EGF to induce phosphorylation. Overexpressionof erbB-2 receptor resulted in a high level of auto-phosphorylation inthese cells. As shown in FIG. 6, lead compound B17 selectively inhibitsthe EGF-induced erbB-2 kinase activity in the EGFR/erbB-2 chimericreceptor of 3T3-EGFR/erbB-2 cells, but not the EGFR kinase activity in3T3-EGFR cells. B17 also inhibits the autophosphorylation of erbB-2 in3T3/erbB-2 cells.

[0070] To test if B17 blocks the MAP kinase activity mediated by erbB-2,we tested the MAP linase activity induced by Heregulin in MDA-453 andinduced by EGF in MDA-468. We found that the MAP kinase activity inMDA-453 was inhibited by B17 with a potency similar to the inhibition ofphosphorylation of erbB-2, while the MAP kinase activity in MDA-468 wasnot inhibited with concentrations of up to 400_M, indicating that B17specifically inhibits the erbB-2 receptor mediated MAP kinase signalingpathway.

[0071] Furthermore, we tested the ability of B17 to inhibit cell growthusing MDA-453, 3T3/erbB-2. MDA-468 and 3T3/EGFR. As shown in FIG. 7, itwas found that B17 exhibits an IC₅₀≦0.625_M in MDA-453 and (3T3/erbB-2data not shown), while only having 25% inhibition at 5_M in MDA-468(FIG. 4) as shown in FIG. 8 and in NIH3T3/EGFR (data not shown).

[0072]FIG. 7 shows the in vivo inhibition of erbB-2 tyrosinephosphorylation in BT-474 cells, which also overexpress the erbB-2 as inMDA-453 cells, but are highly tumorigenic in nude mice. The figure showsthat in tumor-bearing mice (BT-474), there is more than 70% (⅔)reduction in erbB-2 phosphorylation activity compared with the activityobtained for control untreated tumors. The activity of the B17 compoundin animal systems was further probed. FIG. 8 shows that down-streameffector protein, MAP kinase phosphorylation is markedly reduced by invivo treatment of B17 in tumor cells. As shown in FIG. 8, no change inthe expression level of Cerb -2 protein was observed in human breastcarcinoma BT-974 cells 24 hours after intraperitonial injection 100mg/kg of B17.

[0073] Longterm in vivo efficacy investigations were conducted on micetreated with 100 mg/kg of B17, by intraperitonial injection twice perweek. As shown in FIG. 9, the treatment produced about 28% reduction oftumor volume compared to controls at day 15.

[0074] The above results indicate that lead compound B17 is a fairlypotent ATP competitive kinase inhibitor which selectively blocks theerbB-2 kinase activity, thereby shutting-down erbB-2 mediated signalingtransduction pathway. When added directly to cells in culture, it wasfound that B17 inhibits cellular proliferation of erbB-2 overexpressingcells. In addition, we found that B17 is an irreversible inhibitor.

[0075] Based on the unexpected biological activity and selectivity foundfor the compound B17, the compound databases were further searched toidentify analogs of B17 that may have even greater activity and/orselectivity in inhibiting erbB-2 activity. The search in the NCIdatabase produced over 40 closely related analogues of lead compoundB17.

[0076] The following is a table showing the chemical structure andactivity of some of the analogues of B17:

[0077] Rotational Drug Design of erbB-2 Kinase Inhibitors

[0078] In another aspect, the present invention provides novel compoundswhich are rationally designed to inhibit erbB-2 kinase activity.Rational design of the novel compounds is based on information relatingto the binding site of the erbB-2 kinase protein. The structures of theprotein and a lead compound is analyzed such that compound structureshaving possible activity in binding to the binding site are formulated.

[0079] The structure of the lead compounds is divided into design blocksthe modification of which is probed for influence on the interactionsbetween the lead compound and the active site. Compounds havingdifferent design block combinations are then synthesized and theiractivity in relation to the identified mechanism is tested. Such testsare conducted in vitro and/or in vivo, in the same manner describedabove for lead compound B17. The information obtained through such testsis then incorporated in a new cycle of rational drug design. Thedesign-synthesis-testing cycle is repeated until a lead compound havingthe desired properties is identified. The lead compound is thenclinically tested.

[0080] As discussed above in connection with the modeling of thestructure of the erbB-2 kinase domains, it has been found that erbB-2and EGFR have a very similar ATP binding site as compared to otherreceptor kinases such as insulin receptor tyrosine kinase. However, theerbB-2 kinase domain has two distinctive residues (Cys805 and Ser753)located at the ends of the ATP binding site (FIG. 1). The Cys805 iscommon within the EGFR family but not shared by other receptor kinases.Ser783 is unique to erbB-2 (EGFR has a Cys residue at this position).

[0081] The distinguishing structural features of the erbB2 receptor sitewere employed as a guide in rationally designing novel compounds havingenhanced binding activity and selectivity towards the erbB-2 receptor.The structure of lead compound B17 was rationally modified to enhancethe interactions between B17 and the Ser 780.

[0082] Based on the above computational modeling, database searching,rational drug design, in vitro and in vivo biological testing, thepresent inventors have discovered that compounds having the genericformula set forth below specifically interact with erbB-2 kinasemolecules:

[0083] Thus, the compounds produced according to the invention will beused to treat conditions wherein inhibition of erbB-2 kinase signalingis therapeutically beneficial. This will include conditions that involveabnormal cell growth and/or differentiation such as cancers and otherneoplastic conditions. Also, the subject compounds may be used to treatother conditions involving abnormal cell proliferation and/ordifferentiation such as dermatological conditions and disorders. Also,the subject compounds may be useful in treating inflammatory conditionssuch as arthritis, psoriasis, autoimmune disorders such as myastheniagravis, lupus, multiple sclerosis, and others, and conditions involvingabnormal platelet aggregation. The preferred indication is cancer,especially cancers involving over-expression of erbB-2 EGF and/or thePDGF receptor, cancers that express mutant ras, or cancers whichcomprise a Bcr/Abl translocation. Examples of cancers which may betreated according to the invention include breast colon, pancreatic,prostate, head and neck, gastric, renal, brain and CML.

[0084] The subject therapies will comprise administration of at leastone compound according to the invention in an amount sufficient toelicit a therapeutic response, e.g., inhibition of tumor cellproliferation and/or differentiation and/or promotion of apoptosis.

[0085] The compound may be administered by any pharmaceuticallyacceptable means, by either systemic or local administration. Suitablemodes of administration include oral, dermal, e.g., via transdermalpatch, inhalation, via infusion, intranasal, rectal, vaginal, topicalparenteral (e.g., via intraperitoneal, intravenous, intramuscular,subcutaneous, injection).

[0086] Typically, oral administration or administration via injection ispreferred. The subject compounds may be administered in a single dosageor chronically dependent upon the particular disease, condition ofpatient, toxicity of compound, and whether this compound is beingutilized alone or in combination with other therapies. Chronic orrepeated administration will likely be preferred based on otherchemotherapies.

[0087] The subject compounds will be administered in a pharmaceuticallyacceptable formulation or composition. Examples of such formulationsinclude injectable solutions, tablets, milk, or suspensions, creams,oil-in-water and water-in-oil emulsions, microcapsules andmicrovesicles.

[0088] These compositions will comprise conventional pharmaceuticalexcipients and carriers typically used in drug formulations, e.g.,water, saline solutions, such as phosphate buffered saline, buffers,surfactants.

[0089] The subject compounds may be free or entrapped in microcapsules,in colloidal drug delivery systems such as liposomes, microemulsions,and macroemulsions. Suitable materials and methods for preparingpharmaceutical formulations are disclosed in Remington's PharmaceuticalChemistry, 16 Edition, (1980). Also, solid formulations containing thesubject compounds, such as tablets, and capsule formulations, may beprepared.

[0090] Suitable examples thereof include semipermeable materials ofsolid hydrophobic polymers containing the subject compound which may bein the form of shaped articles, e.g., films or microcapsules, as well asvarious other polymers and copolymers known in the art.

[0091] The dosage effective amount of compounds according to theinvention will vary depending upon factors including the particularcompound, toxicity, and inhibitory activity, the condition treated, andwhether the compound is administered alone or with other therapies.Typically a dosage effective amount will range from about 0.0001 mg/kgto 1500 mg/kg, more preferably 1 to 1000 mg/kg, more preferably fromabout 1 to 150 mg/kg of body weight, and most preferably about 50 to 100mg/kg of body weight.

[0092] The subjects treated will typically comprise mammals and mostpreferably will be human subjects, e.g., human cancer subjects.

[0093] The compounds of the invention may be used alone or incombination. Additionally, the treated compounds may be utilized withother types of treatments, e.g., cancer treatments. For example, thesubject compounds may be used with other chemotherapies, e.g.,tamoxifen, taxol, methothrexate, biologicals, such as antibodies, growthfactors, lymphokines, or radiation, etc. Combination therapies mayresult in synergistic results. In particular, the compounds may beadvantageously used in conjunction with herceptin based therapies.

[0094] The preferred indication is cancer, especially the cancersidentified previously While the invention has been described in terms ofpreferred embodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

What is claimed is:
 1. A method of therapy which results in theinhibition of erbB-2 kinase in a subject in need of such inhibitionwhich comprises administering a therapeutically effective amount of atleast one compound having one of generic formulae (I) to (V) or apharmaceutically acceptable salt thereof:

wherein R₁ is a substituted monocyclic or bicyclic aryl or heteroaryland R₂ is selected from hydrogen, alkyl, and heteroaryl.
 2. The methodof claim 1, wherein R₁ is benzene, pyidone, pyrasine, pyrimidine,pyridazine, naphtalene, naphthyridine, benzofuran, benzothiophene,indole, 1H-indazole, indoline, benzopyrazole, 1,3-benzodioxole,quinazoline, pyridopyrazine, pyridopyridine, quinolone, benzothiazole,quinoxaline, benzoxazine, phtalazine or cinnoline.
 3. The method ofclaim 1, wherein said inhibiton antagonizes erbB-2 cell growthsignaling.
 4. The method of claim 1, wherein said method results in theinhibition of cell proliferation and/or differentiation.
 5. The methodof claim 1, which results in the induction of apoptosis.
 6. The methodof claim 1, which results in treating cancer.
 7. The method of claim 6,wherein said cancer is one that is characterized by cells thatover-express the erbB-2 and/or the EGF receptor, cells that express amutant ras, or cells that comprise a Bcr/Abl transfection.
 8. The methodof claim 6, wherein the cancer is selected from the group consisting ofbreast, colon, pancreatic, prostate, head and neck, gastric, renal,brain and CML.
 9. The method of claim 1, wherein said compound isadministered by a method selected from the group consisting of oral,intranasal, intraperitoneal, intravenous, intramuscular, intratumoral,rectal, and transdermal.
 10. The method of claim 1, wherein theadministered amount of said compound ranges from 1 mg to 1 g/kg of theweight of said subject.
 11. The method of 10, wherein the amount of saidcompound more preferably ranges from 5 mg to 50 mg/kg of the weight ofsaid subject.
 12. The method of claim 6, which further comprises theadministration of another anticancer compound, radiation, or a compoundthat induces apoptosis.
 13. The method of claim 6, which furthercomprises the administration of herceptin.
 14. A method of inhibitingcell growth in a subject in need of such inhibition comprisingadministering to a subject an effective amount of at least one compoundhaving one of generic formulae (I) to (V) or a pharmaceuticallyacceptable salt thereof:

wherein R₁ is a substituted monocyclic or bicyclic aryl or heteroaryland R₂ is selected from hydrogen, alkyl, and heteroaryl one compoundaccording to claim
 1. 15. The method of claim 14, wherein saidinhibition involves inhibiting erbB-2 kinase activity.